PROJECT SUMMARY Human adaptive responses to influenza vaccination and infection are complex, and can be impaired in a variety of conditions, including pregnancy and advanced age, for reasons that are still unclear. We will carry out extensive analyses of lymphocyte phenotypic repertoires, including those of NK cells, and the BCR and TCR sequence repertoires expressed by influenza-specific B cells and T cells, in clinical cohorts designed to include key vulnerable populations, pregnant women and the elderly, known to have impaired responses to influenza vaccination or infection. To evaluate potential specific defects in stages of germinal center reactions, and the proliferation and selection of influenza-specific lymphocytes that eventually become detectable in the blood, we will carry out serial fine-needle aspirations (FNAs) from draining lymph nodes after vaccination, and in tonsils from individuals receiving intranasal vaccine. Paired lymph node and blood data should enable detection of defects in responses that have previously not been accessible to study in humans. We will use this approach to identify key changes in immune responses with adjuvant (MF59) in influenza vaccination for the elderly, and correlate these with resultant antibody quality. An additional goal of this Project will be to leverage prior extensive influenza-specific TCR and BCR sequencing efforts in the Davis, Boyd and Robinson labs, as well as public data, to assemble databases of confirmed influenza-specific receptor sequences to test specific hypotheses. With new computational approaches, we will identify and validate convergent or public receptors that share sequence features indicating that they bind similar epitopes. We will test whether the frequency, diversity, or particular epitope targets of such receptor sequences can predict vaccine responses, and, more importantly, protection against live influenza viral challenge. Two different influenza viral challenge cohorts will be used to validate our TCR and BCR predictors of vaccine protection. This project will provide better understanding of age-related and pregnancy-related alterations in influenza vaccine responses, as well as new knowledge about vaccine responses in secondary lymphoid tissues including lymph nodes and tonsils. In the longer term, working within this U19, Project 2 will contribute to global efforts to improve quantitative and predictive understanding of the human immune system. This knowledge should help to shape strategies for improving and testing the next generation of influenza vaccines to prevent future epidemics and pandemics. Many of these new approaches may also provide a template for developing more effective vaccines in general.